present study was conducted at Fish and Fisheries Laboratory, Department of
Zoology, Kurukshetra University, Kurukshetra (29°58’N latitude and 76°51’E
longitude), Haryana, India during 2014-2017.
3.1 Selection of fish species
introduction of alien species has been considered as one of the probable cause
for the extinction of native species. African
Sharptooth Catfish, Clarias gariepnus, (Burchell, 1822) an exotic species
was selected for present study (Fig. 4). The fish has been recently
recognized in abundance in rivers of Indian sub-continent, particularly in
river Yamuna. Thus, remarkable occurrence of C. gariepinus in the river system has made a requisite to
understand the perpetuation of this species.
of the selected fish species
gariepinus (Burchell, 1822), commonly called as mangur or thai mangur,
selected for the present study belongs to catfish family. It is named Clarias which in Greek means ‘lively’
i.e. can survive for long time out of water and gariepinus in reference to its native locality i.e. the Gariep River,
South Africa. In India, it was first introduced from
Thailand through Bangladesh.
Species: gariepinus (Burchell,
It is recognized by its cylindrical body
with scaleless skin, flattened bony head, snout
broadly rounded; eyes supero-lateral and relatively small (Teugels, 1986), elongated spineless dorsal fin reaching upto the base
of caudal fin, narrow occipital process, pectoral fins with bony spines
serrated only on its outer margin and four pairs of barbels around a broad
mouth. Maxillary barbel is quite long but not reaching dorsal fin origin; nasal
and inner mandibular barbels are unequal in length, outer mandibular barbel longer than inner pair (van Oijen, 1995). Distance between
occipital process and base of dorsal fin is short. The anal, caudal and dorsal fins are not united. It
possess large air-breathing labyrinthic organ
arising from gill arches which enables it to breathe under dry conditions. The
sex of the fish can be easily identified on the basis of distinct sexual
papilla (elongated in males and round button-head in females) located
immediately behind the anal opening. 3.1.2
Collection of fish
specimens of Clarias gariepinus in range of 160-1900g body
weight and 26.5-67.5 cm length were
procured monthly throughout the study period from April 2014-March 2017 from mainly
two landing stations of river Yamuna in Delhi region i.e. Wazirabad barrage and
Okhla barrage. Fishes were collected from selected sites with the help of local
fisherman using different types of nets namely cast nets and drag nets. The
specimens were brought to the laboratory in oxygen filled bags and their sex
was differentiated. The fishes were anesthetized with chloroform and their
total length and total weight was recorded for length-weight relationship and immediately
dissected for the removal of gonads for further analysis.
To study water quality,
samples were collected seasonally from landing station of river Yamuna and were
brought to laboratory in polythene bottles for analysis. Various water quality
parameters were analyzed according to standard procedures (APHA, 1998) within
3-4 days of sampling. Methodology and instruments used for analysis of
physicochemical parameters are given in Table 1.
3.3 Length-Weight Relationship (Le Cren, 1951)
of 325 specimens (185 females and 140 males) were sampled from
April 2014-March 2017 in order to calculate the length-weight
relationship and the Fulton’s condition factor. Total length (cm) and body weight
(gm) were recorded using vernier calipers (nearest to 0.1 cm) and digital
weighing balance (nearest to 0.1 gm) respectively. On the
basis of gross morphology of gonads, whole reproductive cycle of fish was
categorized into different maturity stages following CEK and YILMAZ (2007) and Yalcin et al., (2001).
length-weight relationship was calculated by the least square method applying
the Le Cren (1951) formula: W = a Lb. The log transformed data of
the total length and total weight gives a straight line relationship
log W =
log a + b log L
= Weight (g), L = Length (cm), ‘a’ and ‘b’ are Constant
constants ‘a’ represents the point at which the regression line intercepts the
y- axis and ‘b’ the slope of the regression line which were estimated by the
method of least square (Snedecor and Cochran, 1967) and the linear equation was
fitted separately for male and female at different maturity stages.
The co-efficient of determination (r2) was used as an indicator of
the quality of the linear regression provided by the value of ‘b’. The coefficient
of correlation (r) was calculated to study the relationship between length and
factor of a fish indicates the well-being of the fish in a particular
environment. It was assessed for comparisons among different sex, maturity
stages and size groups. It can be numerically determined by
using the formula of Fulton (1904)
Where, K= Condition factor, W
= Weight (g), L = Length (cm)
Fecundity estimation (Doha
and Hye, 1970)
To evaluate the fecundity of the fish,
ripe ovaries from the fish specimens ranging from 32-60.5 cm in length and 90-1700g in weight were utilized. Gravimetric method was
utilized where the external connective tissues and moisture of the ovary were
removed and weight of ovary of each fish was recorded with the help of electronic
balance (Afcoset FX-300). The paired ovaries were kept in Gilson’s fluid for 3 weeks for
hardening of the eggs (Bagenal and Braum, 1978). Sub-sample of 1g from each ovary was
taken; the number of eggs for each portion were sorted out and counted. Number
of eggs in 1g multiplied by the total weight of the ovary gave the total number
of eggs in ovary i.e., the fecundity of respective fish.
3.5 Age analysis (Yalcin et al.,
For the calculation of age, trunk vertebrae were removed and washed
thoroughly for about 2-3 minutes in boiling water to remove the muscles. The
processed vertebrae were sun-dried for 24h,
preserved dry in different plastic envelops and were carefully tagged with
reference number of the fish, date of collection and maturity stage for future
examination. The processed vertebrae were examined for age
determination in xylol and photographed under the Magnius Sterio microscope.
determine the ovum diameter, 20 ovaries from each maturity stage were fixed in
a 10% formalin solution. Subsamples were taken from the anterior, middle, and
posterior regions of the ovary. Diameter of randomly 30 oocytes from each
sub-sample was recorded using ocular micrometer (Erma Inc, Japan).
Studies (Pearse, 1968)
Acid: 75 parts
Formaldehyde: 25 parts
Acetic Acid: 5
Gonads (ovary and testis) were carefully
removed from the dissected fishes and washed in 0.9% NaCl to make them blood
free, cut into small pieces and fixed in Bouin’s fixative for 24 hours.
Tissue was kept overnight for washing under the
running tap water.
Dehydrated in graded series of ethanol (30%–100%) for about 20 minutes in
passed through a mixture of absolute alcohol and xylene (1:1) for about 10
minutes and finally in pure xylene for 6-8 minutes.
For embedding, the cleared tissue were passed
through mixture of xylene and paraffin wax (1:1), followed by transfer in pure
wax for 12 hours at 60°C.
Blocks were prepared by placing the tissues in
proper orientation in the suitable sized cavity block filled with molten wax
and allowed to solidify.
The embedded blocks were trimmed and sectioned serially at 5 µm on a
microtome (Weswox Optik Model MT-1090A 15125).
The thin sections were stretched on albumin
coated slides and left in oven (37°C)
The sections were de-paraffinized in xylene and
were brought down to water after downgrading through series of alcohol (100-30%);
subsequently stained with hematoxylin for 5-8 min and passed through tap water
until color developed.
The slides were passed into upgraded series of
alcohol (30-70%), stained in eosin for 2 minutes and further dehydrated through
90% and 100% alcohol.
Sections were cleared with xylene and mounted
The slides were observed under Olympus CX41 light
microscope. Microphotographs were taken with the help of C-7070, Olympus
3.8 Ultrastructural Studies
3.8.1 Transmission Electron Microscopy (Bancroft
and Stevens, 1982)
g toluidine blue dissolved in 100.0 mL distilled water
Uranyl acetate: excess of uranyl acetate
to 10ml of filtered 50%
ethanol in centrifuge tube.
citrate: one half of pellet of sodium hydroxide to 12ml of double distilled
water; add 50mg of lead citrate, mix and centrifuge.
Araldite CY212- 10 ml
Dodecenyl Succinate Anhydride
(DDSA)- 10 ml
2,4,6 tri (dimethyl amino methyl) phenol-30
(DMP-30)- 0.4 ml
Plasticizer (dibutyl phthalate) –
1. Transverse sections were fixed for 24 hours in Karnovsky’s
fixative in 0.2M phosphate buffers saline at 7.2 pH.
2. After fixation, specimens were rinsed
thoroughly in 0.1 M phosphate buffer to wash off the excess fixative.
3. Tissue were trimmed to appropriate size (1mm),
and post-fixed in 1% osmium tetra-oxide for 1 hour at 4°C and subsequently rinsed thoroughly in buffer to wash off excess
4. Fixed tissues were dehydrated in graded acetone
series (30-100%) for 15 minutes each; cleared in toluene for 30 minutes and
infiltrated with resin.
5. Processed tissues were embedded in pure
embedding medium using flat moulds with paper label written with pencil put
along the sidewall of the capsule.
6. Moulds were baked at 50°C for overnight, then at 60°C, until the resin gets hardened.
After polymerization blocks were removed by bending the mold sideways or by
cutting the capsules with a razor blade.
7. Blocks were trimmed manually to produce a pyramidal shape at the
tip of the block where the tissue is present.
8. 1?m thick sections were cut using glass knives
(prepared using Belgium glass strips) in a porter-blum MT II ultracutE and stained with toluidene blue,
washed in running water followed by distilled water, dried and mounted with
DPX. Sections were examined under light microscope to determine the section
9. Ultra-thin sections were prepared with the help
of fresh glass knives, mounted on 100 mesh grids and stained with uranyl
acetate and lead citrate.
10. Sections were photographed under Morgagni 268 D
TEM at All India Institute of Medical Sciences, New Delhi.